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Protein and Cell

, Volume 8, Issue 5, pp 379–393

First Online: 23 January 2017Received: 13 September 2016Accepted: 01 December 2016DOI: 10.1007-s13238-016-0360-8

Cite this article as: Guo, J., Ma, D., Huang, R. et al. Protein Cell 2017 8: 379. doi:10.1007-s13238-016-0360-8 ABSTRACT

Human pluripotent stem cells hPSCs are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline Dox inducible dCas9-VP64-p65-Rta dCas9-VPR transcription activator and a reverse Tet transactivator rtTA expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass ICM of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

KeywordsCRISPR transcription activation human pluripotent stem cells NANOG pluripotency Electronic supplementary materialThe online version of this article doi:10.1007-s13238-016-0360-8 contains supplementary material, which is available to authorized users.





Autor: Jianying Guo - Dacheng Ma - Rujin Huang - Jia Ming - Min Ye - Kehkooi Kee - Zhen Xie - Jie Na

Fuente: https://link.springer.com/







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